Enteroviruses belong to the family Picornaviridae that represents a very large RNA-virus family with respect to the number of members but one of the smallest in terms of virion size and complexity. The virion of an enterovirus consists of a capsid of 60 subunits. Each subunit consisting of the four proteins (VP 1-VP4) arranged with icosahedral symmetry around a genome made up of a single strand of positive-sense RNA. Enteroviruses are transient inhabitants of the human alimentary tract and may be isolated from the lower intestine or the throat. Enteroviruses of human origin include polioviruses (serotypes 1 to 3), coxsackieviruses of groups A (CAV, serotypes 1 to 22 and 24) and B (CBV, serotypes 1 to 6), echoviruses (serotypes 1 to 9, 11 to 27 and 29 to 33) and enteroviruses (serotypes 68-71). Enteroviral infections in humans may result in a wide range of acute symptoms involving neurological, skin and mucosa, cardiac and muscular, ocular, respiratory and gastrointestinal conditions, as well as undifferentiated febrile illness, generalized diseases of infants and diabetes mellitus. Among the nonpolio enteroviruses, some unsubclassified serotypes have been particularly prevalent in some countries and areas during summer and early fall, e.g., enteroviruses 70 and 71. Enterovirus 71 has been isolated from patients with meningitis, encephalitis and paralysis resembling poliomyelitis. It continues to be one of the main causes of central nervous system diseases, sometimes fetal, around the world. Furthermore, the virus has caused outbreaks of human hand-foot-and-mouth disease in some areas, e.g., Japan, Sweden and Taiwan.
Common diagnoses of enteroviruses usually include recovery of the virus and serological tests. The recovery of the virus may includes isolation of the viruses from throat washings, throat swabs, stools, rectal swabs and sometimes cerebrospinal fluid (in aseptic meningitis cases). The isolated viral specimens are inoculated into tissue cultures or suckling mice (the latter is specific for coxsackieviruses) for propagation and identification. It takes one to two weeks to complete a run of viral recovery. In addition, intervention has to be made by qualified technicians.
Serologic tests are generally conducted by means of neutralization assays detecting the neutralizing antibodies specific to the infecting virus when they exist. For some echoviruses, hemagglutination-inhibition assays might show type-specific infections. Serum antibodies can also be detected or titrated by the immunofluorescence technique using infected cell cultures on coverslips as antigens. However, serologic tests are difficult to evaluate because of the multiplicity of serotypes, unless the antigen used has been isolated from a specific human or during an epidemic outbreak of typical clinic illness. In all of the serologic assays, skilled artisans are required to judge the cut-off values from the readouts. Also, a period of time and specialized techniques are usually required to prepare such an antigen used in those assays to reduce the incidence of heterotypic reactions or cross-reactions.
Neither the recovery of the virus nor the serologic tests provides a rapid diagnostic tool. Nevertheless, the acute syndromes associated with enteroviruses may progress fast, and in some cases may even become fatal in a couple of days. There is therefore a need for a method to rapidly detect enteroviruses with ease in practice.
The present invention relates to novel pairs of oligonucleotide primers for use in detecting the presence or absence of an enterovirus in a sample. Each pair of primers according to the present invention consists of a first primer and a second primer, which are useful in rapidly diagnosing the diseases or conditions associated with enteroviruses by nucleic acid amplification assays, such as polymerase chain reactions.
It is surprisingly found in the present invention that some nucleotide sequences correspond to the conserved portions in the nucleic acids of the enteroviruses. Therefore, the present invention further provides synthetic nucleotide sequences capable of specifically hybridizing to a sense strand of an enterovirus nucleic acid or a nucleic acid corresponding to the sense strand, such as the product of an amplification reaction with the sense strand as the template.
In another aspect, the present invention relates to a method of detecting the presence or absence of an enterovirus nucleic acid in a sample. The method according to the present invention comprises (a) contacting the sample with a pair of oligonucleotide primers according to the present invention in an amplification process; and (b) determining the presence or absence of an enterovirus by detecting the presence or absence of amplification products.
Preferably, in the method according to the present invention, the sample may be simultaneously contacted with a second pair of oligonucleotide primers according to the present invention in an amplification process in addition to the first pair of primers. The primers in the second pair are not the same as the primers in first pair. The primers in the second pair may have either the first or the second primer different from the corresponding primer in the first pair. Preferably, both primers in the second pair are different from the ones in the first pair. Furthermore, the sample may be simultaneously contacted with a third pair of the oligonucleotide primers according to the present invention in an amplification process, in addition to the first and second pairs of primers. In a similar manner, the third pair of primers is neither the same as the first pair of primers nor the second pair of primers.
In an alternative manner, the method according to the present invention may further comprise contacting the products of step (a) with a second pair of the oligonucleotide primers according to the present invention in an amplification process. The second pair of primers is chosen so as to be capable of being used to amplify a sequence in an amplification process that is equal to or within the sequence obtainable in the amplification process using the first pair of primers.
In another alternative of the method according to present invention, the amplification products detected in step (b) are further subjected to a specific hybridization with at least one synthetic nucleotide sequence according to the present invention.
In yet another aspect, the present invention relates to a method of detecting and differentiating enterovirus type 71 in a sample. The method comprises (a) contacting the sample with at least one pair of oligonucleotide primers according to the present invention in an amplification process; (b) determining the presence or absence of enterovirus type 71 by detecting for the presence or absence of amplification products; and (c) subjecting the amplification products detected to a specific hybridization with at least one synthetic nucleotide sequence comprising the sequence of SEQ ID NO: 12 or SEQ ID NO:13. Preferably, the amplification products detected are subjected to a specific hybridization with the synthetic nucleotide sequences comprising the sequences of SEQ ID NO:12 and SEQ ID NO:13.
In yet another aspect, the present invention relates to a method of detecting and differentiating coxsackievirus A16 in a sample. The method comprises contacting the sample with a pair of oligonucleotide primers according to the present invention in an amplification process; determining the presence or absence of coxsackievirus A16 by detecting the presence or absence of amplification products; and subjecting the amplification products detected to a specific hybridization with at least one synthetic nucleotide sequence comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15. Preferably, the amplification products detected are subjected to a specific hybridization with the synthetic nucleotide sequences comprising the sequences of SEQ ID NO: 14 and SEQ ID NO: 15.
In yet another aspect, the present invention relates to a method of detecting and differentiating enterovirus type 71 and/or coxsackievirus A16 in a sample. The method comprises contacting the sample with a pair of oligonucleotide primers according to the present invention in an amplification process; determining the presence or absence of enterovirus type 71 and/or coxsackievirus A16 by detecting the presence or absence of amplification products; and subjecting the amplification products detected to a specific hybridization with at least a first synthetic nucleotide sequence comprising the sequence of SEQ ID NO:12 or SEQ ID NO: 13, and with at least a second synthetic nucleotide sequence comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15. Preferably, the amplification products detected are subjected to a specific hybridization with the synthetic nucleotide sequences comprising the sequences of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.
In yet another aspect, the present invention provides a kit for detecting an enterovirus in a sample, which comprises at least one pair of oligonucleotide primers according to the present invention. Preferably, the kit according to the present invention comprises more than one pair of primers according to the present invention. In an alternative of the present invention, the kit may further comprise at least one synthetic nucleotide sequence according to the present invention.
In yet another aspect, the present invention provides a kit for detecting and differentiating enterovirus type 71 in a sample, which comprises at least one pair of oligonucleotide primers according to the present invention and at least one synthetic nucleotide sequence comprising the sequence of SEQ ID NO: 12 or SEQ ID NO: 13. Preferably, the kit according to the present invention comprises more than one pair of primers according to the present invention. In a preferred embodiment of this invention, the kit comprises the synthetic nucleotide sequences comprising the sequences of SEQ ID NO: 12 and SEQ ID NO:13.
In yet another aspect, the present invention provides a kit for detecting and differentiating coxsackievirus A16 in a sample, which comprises at least one pair of oligonucleotide primers according to the present invention and at least one synthetic nucleotide sequence comprising the sequence of SEQ ID NO: 14 or SEQ ID NO: 15. Preferably, the kit according to the present invention comprises more than one pair of primers according to the present invention. In a preferred embodiment of this invention, the kit comprises the synthetic nucleotide sequences comprising the sequences of SEQ ID NO:14 and SEQ ID NO:15.
In yet another aspect, the present invention provides a kit for detecting and differentiating enterovirus type 71 and/or coxsackievirus A16 in a sample, which comprises at least one pair of oligonucleotide primers according to the present invention; and at least a first synthetic nucleotide sequence comprising the sequence of SEQ ID NO:12 or SEQ ID NO:13 and at least a second synthetic nucleotide sequence comprising the sequence of SEQ ID NO:14 or SEQ ID NO:15. Preferably, the kit according to the present invention comprises more than one pair of primers according to the present invention. In a preferred embodiment of this invention, the kit comprises the synthetic nucleotide sequences comprising the sequences of SEQ ID NOs:12-15.